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WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6
BOIL 3/00, G01N 33/543

(11) International Publication Number: WO 98/15356

(43) International Publication Date: 16 April 1998 (16.04.98)

(21) International Application Number: PCT/GB 97/02708

(22) International Filing Date: 8 October 1997 (08.10.97)

(30) Priority Data:

9620934.1

8 October 1996 (08.10.96)

(71) Applicant (for all designated Slates except US): MOLECULAR

DRIVES LIMITED [GB/GB]; University of Glasgow, 2 The
Square, Glasgow G12 8QQ (GB).

(72) Inventor; and

(75) Inventor/Applicant (for US only): GORDON, John, Francis
[GB/GB]; 5 Park Crescent, Torrance, Glasgow G64 4BH
(GB).

(74) Agents: McCALLUM, William, Potter et aL; Cruikshank &
Fairweather, 19 Royal Exchange Square, Glasgow Gl 3AE
(GB).

(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
GH, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK,
LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO,
NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR,
TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (GH,
KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ,
BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
ML, MR, NE, SN, TD, TG).

Published

With international search report.

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Title: APPARATUS AND METHOD FOR CONDUCTING ASSAYS

(57) Abstract

A multi-well assay plate structure (54) and assay apparatus and a method for performing chemical biochemical assays is described.
The multi-well assay plate structure (54) defines a relatively shallow substantially enclosed space (71) above a plurality of wells (76), with
the enclosed space (71) having an inlet (72) and an outlet (22) separate from the inlet. Fluid introduced via the inlet (72) flows into the
space (71) and/or wells (76) by displacing air. Withdrawal of the fluid via the inlet (72) or outlet leaves fluid in the wells (76) allowing
various tests to be performed. Various embodiments of the structure are described. The preferred arrangement embodies the structure on
a transparent plastic disk which can be used with automatic fluid handling apparatus (80) and the results assessed using optical assessment
apparatus (81). The apparatus can be used to perform a variety of assays but, in particular, biochemical/chemical assay, immunoassays and
genetic (DNA) assays and it can be used in a laboratory for multiple sample testing or at a point-of-care, i.e. in a surgery or clinic.

4 r %

FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lesotho

Slovenia

Armenia

Finland

Lithuania

Slovakia

Austria

France

Luxembourg

Senegal

Australia

Gabon

Latvia

Swaziland

Azerbaijan

United Kingdom

Monaco

Chad

Bosnia and Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ghana

Madagascar

Tajikistan

Belgium

Guinea

The former Yugoslav

Turkmenistan

Burkina Faso

Greece

Republic of Macedonia

Turkey

Bulgaria

Hungary

Mali

Trinidad and Tobago

Benin

Ireland

Mongolia

Ukraine

Brazil

Israel

Mauritania

Uganda

Belarus

Iceland

Malawi

United States of America

Canada

Italy

Mexico

Uzbekistan

Central African Republic

Japan

Niger

Viet Nam

Congo

Kenya

Netherlands

Yugoslavia

Switzerland

Kyrgyzstan

Norway

ZVV

Zimbabwe

Cdte d'lvoirc

Democratic People's

New Zealand

Cameroon

Republic of Korea

Poland

China

Republic of Korea

Portugal

Cuba

Kazakstan

Romania

Czech Republic

Saint Lucia

Russian Federation

Germany

Liechtenstein

Sudan

Denmark

Sri Lanka

Sweden

Estonia

Liberia

Singapore

WO 98/15356 PCT/GB97/02708

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APPARATUS AND METHOD FOR CONDUCTING ASSAYS

The present invention relates to apparatus and to a
method for conducting assays and, in particular, to multi-
well plate structures for receiving and holding, in
separate wells, volumes of liquid for the purpose of
5 conducting chemical or biochemical assays. Mult i -well

trays or plates having a 2 -dimensional array of small wells
are commonly used in medicine and science to facilitate
testing of a liquid analyte . One particular area of use is
blood screening where blood or blood products are

10 introduced into the wells to test for viruses such as HIV,

hepatitis etc.

Such tests (immunoassays ) typically involve an
antigen-antibody interaction, where the surfaces of the
wells are coated with specific antigen itself. This

15 approach detects circulating antibodies to that specific

antigen. Alternat ively the wells can be coated with a
specific antibody which captures circulating antigen which
is, in turn, identified by a second antibody directed
against a second epitope on the captured antigen. These

2 0 two approaches are just two of the large number of variants

developed in immunoassay (review Principles and Practice of
Immunoassay Price & Newman 1997 ISBN 1-56159-145-0) .

In an immunoassays sample must be applied and in most
cases subsequent addition of reagents or washing buffer is

25 required. Typically the well is exposed to blood or blood

product and the well is rinsed clean and a further
reactant, which binds either to exposed antibodies or
captured antigens is introduced into the wells, to create
an observable reaction. These reactions may produce a

30 colour or some other observable change. This enables the

wells containing specific antigen antibody reactions to be
identified and the extent of these reactions quantified.

It is often necessary to fill each well of a multi-
well tray with a precisely defined volume of analyte. This

3 5 is normally achieved using a single or mult i -headed micro-

WO 98/15356 PCT/GB97/02708

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pipette. However, this process is often time consuming
and, particularly where a large number of wells are to be
filled, can lead to a number of wells being missed.

In certain circumstances it is necessary that the
5 wells of a tray be contained within a substantially closed

container, e.g. to avoid the risk of contamination of the
wells and of leakage of contaminated material. With trays
such as this, it may be difficult or impossible to gain
access to the wells to enable them to be filled using a
10 micro-pipette.

It is an object of the present invention to overcome
or at least mitigate the disadvantages of known multi-well
trays .

This is achieved by providing a multi-well assay plate
15 structure which defines a relatively shallow substantially

enclosed space above a plurality of wells, with the
enclosed space having an inlet and an outlet separate from
the inlet. Fluid introduced via the inlet flows into the
space, and covers the wells, by displacing air.
20 Withdrawal of the fluid from the space via the inlet or

outlet leaves fluid in the wells allowing various tests to
be performed.

According to a first aspect of the present invention
there is defined a multi-well assay plate structure
25 comprising:

a first upper surface,

a second lower surface having a plurality of wells
disposed therein,

the first and second surfaces defining a chamber
30 having an inlet and an outlet, the inlet and outlet

allowing fluid to be introduced and withdrawn from the
chamber, the wells being proportioned and dimensioned to
retain a volume of fluid in each well following withdrawal
of the liquid.

3 5 Preferably, the chamber is shallow enough to allow

fluid to fill the wells and the chamber. The wells are
deep enough to retain a volume of fluid following

WO 98/15356 PCT/GB97/02708

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withdrawal of fluid in the space above the wells.

The plate structure can be of any convenient shape
but, advantageously, is sector-shaped with a detachable
handle at the longer arc-portion to facilitate locating the
5 sector on a disc. Conveniently, a plurality of sector-

shaped structures are located on the disc.

Conveniently, also the sectors and discs are made of
plastic and the sectors can be snap- fitted onto the disc.
The sectors and the disc include lock and key portions to
10 allow the sectors to be snap- fitted in the correct

orientation only.

Alternatively, a disc with a plurality of separate
sections can be manufactured or moulded in one piece
instead of snap-in sectors.
15 The composite structure may be snap- fitted onto a

compact disk.

The disk structure may have a circumferential gutter
extending around its periphery to facilitate collection of
fluid following fluid introduction/withdrawal from the
2 0 chamber.

The wells are dimensioned and proportioned in terms of
diameter and depth to receive and retain fluid containing
the analyte or part of the reagent under test. The exact
dimensions are a matter of choice and depend on a number of
25 parameters such as the type of material of the surfaces of

the chamber and wells; viscosity of the fluid and the depth
(height) of the space between the first and second
surfaces .

Advantageously, the dimensions of the structure are
30 such that the wells fill to retain sufficient fluid the

space is flooded and withdrawal to allow a measurable
reaction to be measured within an individual well without
contribution from adjacent wells. The overall process of
sequential steps of flood and fill is advantageous in that
35 it allows both discrete measurements within individual

wells when filled and efficient washing of an array of
wells (flood) which is useful in multistep procedures, such

WO 98/15356 PCT/GB97/02708

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as immunoassays, which requires sequential application of
reagents interspersed with rigorous washing steps. This
permits the wells to be cleaned or rinsed in the same way
as filling to allow subsequent tests to be carried out
5 within an individual well whilst avoiding cross-

contamination between adjacent wells.

The structure is preferably made of transparent or
otherwise optically transmissive plastic to facilitate
optical reading of the wells to determine the results of

10 the tests. Conveniently, the structure is integrated with

automatic fluid handling apparatus and an optical reader to
allow automatic fluid handling and optical assessment of
the results of the reactions. Alternatively, fluid

handling can be manually controlled and the results of the

15 reactions within the structure can be assessed by an

optical reader or be scored by visual assessment.

According to a second aspect of the present invention
there is provided a mult i -well assay structure comprising
an upper surface and a lower closely spaced opposed

20 surface, said upper and lower surfaces defining a

relatively shallow space therebetween, the lower surface
having a plurality of wells therein, at least two spaced
apart openings providing access to said space from an
external location, wherein a fluid introduced into said

25 space through one of said openings fills substantially all

of the space and covers of the wells and said fluid, when
subsequently withdrawn through the same or the other
opening, leaves the wells filled with liquid.

The volume of fluid introduced into each well when

3 0 using the structure of the present invention is

substantially defined by the volume of the well. The
accuracy and precision with which the wells can be filled
is therefore defined by the accuracy and precision with
which the wells can be fabricated and which is generally

35 high. Furthermore, the multiplicity of wells can be filled

by way of a single injection and withdrawal of fluid
through an opening into the space containing the wells, so

WO 98/15356 PCT/GB97/02708

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that the wells can be filled extremely rapidly.

The structure of the present invention provides for
the filling of a plurality of wells in a substantially
closed chamber, the only openings into that container being
5 the fluid injection opening and a second 'vent' opening.

The structure of the present invention simplifies the
process of cleaning or rinsing previously filled wells as
this can be achieved by repeatedly injecting and
withdrawing fluid through one of said openings.
10 Conveniently, the spacing between said upper and lower

surfaces is sufficiently small to facilitate the flow of
fluid in said space by capillary or capillary like action.
Typically, the spacing is less than 1mm and preferably less
than 0 . 5mm .

15 Preferably, said upper and lower surfaces are

substantially planer.

The wells may have any suitable geometry. For
example, the wells may be provided in said lower surface by
blind circular holes with a semi -spherical termination.

20 Alternatively, the wells may have substantially straight

sidewalls, e.g. so that the sidewalls extend substantially
vertically and terminate in a flat base. Vertical
sidewalls assist in preventing the, transfer of fluid
between adjacent wells.

25 The surfaces may be provided by respective upper and

lower plates which are spaced apart by one or more spacer
walls .

Preferably, the opening through which fluid is
introduced into said space is provided through either the
30 upper or lower surface and, more preferably, through the

upper surface. The additional opening may be provided
through said upper or lower surface or through a side
surface .

Preferably, said opening for introducing a fluid
35 comprises a relatively small opening arranged to receive

the end of a syringe or similar liquid injecting device,
where the opening forms a substantially air-tight seal

WO 98/15356 PCT/GB97/02708

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around said end.

Preferably, said lower surface of the container is
treated to increase the hydrophobicity to facilitate smooth
flow of liquid across the sector and hydrophilicity to aid
5 movement of liquid into desired locations, e.g. wells.

This helps to. prevent the formation of air pockets in the
space and aids filling of the wells. The treatment may
comprise for example exposing the surface to a wetting
agent, e.g. poly- 1- lysine , or exposing the surface to a gas

10 plasma.

In one embodiment of the present invention, the multi-
well structure is embodied in a disc. The disc effectively
comprises upper and lower circular plates, the internal
surfaces of which respectively define said upper and lower

15 opposed surfaces. Preferably, said opening for introducing

liquid into the space is a hole passing through the upper
circular plate. Preferably, the second opening is provided
at the peripheral edge of the disc. The space between the
upper and lower plates is subdivided, by one or more

20 dividing walls, to provide a plurality of multi-well plates

in which case each space is provided with an opening and a
vent to enable each space to be independently filled. The
dividing walls may extend radially and/or may be concentric
to one another .

25 Preferably, at least one of the upper and lower plates

forming the container are transparent to enable optical
inspection of the wells from outside the container. The
other of the upper and lower plates may comprise a
reflecting surface so that radiation entering into the

3 0 container through the transparent plate transverses the

container in both directions, resulting in an improved
signal detection for optical inspection.

In an alterative embodiment of the present invention
there is provided a disc arranged to receive a plurality of

35 sector (pie) shaped inserts each of which comprises a

generally planar upper surface having a plurality of wells
provided therein. For each insert, the disc comprises a

WO 98/15356

PCT/GB97/02708

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substantially planar surface arranged, in use, to oppose
said substantially planar insert surface and means for
retaining the insert in position so that the respective
planar surfaces are in closely spaced opposition to one
5 another, and said at least two openings.

Preferably, the opening, for filling the container is
provided through the planar surface of the disc. The vent
opening is preferably provided at, or adjacent to, the
peripheral edge of the disc.

10 The disc preferably comprises upper and lower circular

plates separated by radially extending spacers. The
spacers define slots between the plates for receiving said
inserts. Preferably, said planar surface of each insert
comprises upstanding walls around at least a portion of its

15 periphery for the purpose of sealing the inner edges of the

insert to the opposed planar surface of the disc, thereby
to prevent seepage of liquid around the insert .

According to a third aspect of the present invention
there is provided a method of filling the wells of the

20 multi-well structure of the above first aspect of the

present invention, said method comprising the steps of:

introducing a fluid into said chamber through one of
said openings to substantially flood the chamber;

and subsequently withdrawing the fluid from the

25 chamber through the same or the other opening to leave

liquid in the wells.

Preferably, the method further includes the step of
forming an air tight seal between the fluid inlet and an
end region of a syringe or similar liquid injecting device,

3 0 and injecting fluid through the opening into the chamber

and subsequently sucking liquid out of the space through
the opening.

According to a fourth aspect of the present invention
there is provided a method of conducting a chemical or
35 biochemical assay said method comprising the steps of:

providing a surface within a substantially enclosed
chamber having a plurality of wells at spaced locations

WO 98/15356 PCT/GB97/02708

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sufficient to allow a reaction at each well location,

treating each well with a first reagent, flooding the
enclosed chamber and covering the wells with a fluid
carrying at least a second reagent,
5 removing excess fluid from said chamber to leave a

mixture of said first and second reagents in each well, and

optically assessing each well and determining if a
reaction occurred and correlating the reaction results to
provide an assay of the chemical or biochemical reactions
10 under test.

Preferably, the step of optical assessment is carried
out automatically using optical reading apparatus.

Preferably also, the surfaces with the wells having
first fluid carrying reagents are prior prepared for
15 loading into the structure.

Conveniently, the fluid carrying at least the second
reagent is introduced into the structure and withdrawn from
the structure using suitable automatic fluid handling
apparatus .

20 Conveniently also, after optical assessment of the

results of the assay, the automated fluid handling
apparatus is used to inject and withdraw rinsing fluid a
predetermined number of times from the well tray to clean
the wells for receiving subsequent samples for assay.

25 According to a fifth aspect of the present invention,

there is provided chemical/biochemical assay apparatus
comprising an assay plate structure defined in said first
aspect and having a plurality of wells for receiving
samples to be assayed,

3 0 fluid handling means for introducing and removing

fluid reagents into said assay plate structure to allow a
fluid reagent mixture to be retained in each well, and

optical assessment means for measuring optical result
of the reaction in each well.

35 Preferably, the fluid handling means and the optical

assessment means are automated.

According to a sixth aspect of the present invention

WO 98/15356 PCT/GB97/02708

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there is provided an assay plate structure for use in
conducting optical assays of a fluid analyte, the plate
structure comprising:

a disc for rotation about a central axis, the disc
5 having upper and lower plates and a plurality of

substantially radially extending walls disposed between the
plate, wherein said walls sub-divide the disc into a
plurality of disc sectors; and

a plurality of disc inserts arranged to be received by
10 respective disk sectors and to be retained therein,

the structure further having a plurality of openings
through the upper surface, at least one opening above each
disc sector for introducing a liquid analyte into the
sector space between the plate and the disc insert .
15 Preferably, the disc further comprises a lower plate,

spaced apart from said upper plate by said radially
extending walls. More preferably, the upper and lower
plates are circular.

Preferably, the upper surface of each disc insert and
20 the opposed surface of the plate are substantially planar,

and, more preferably, are in a closely spaced arrangement.

Preferably, a vent opening is provided for each disc
segment around the periphery thereof, between the radially
outer edge of the upper plate and each disc insert .
25 These and other aspects of the present invention will

now be described with reference to the accompanying
drawings, in which:

Fig. 1 is a diagrammatic representation of a multi-
well assay plate structure according to a first embodiment
30 of the present invention;

Figs. 2a to 2c illustrate the steps involved in
filling the wells of the container of Fig. 1;

Fig. 2d is an enlarged detail of part of the structure
of Figs. 2a to 2c;
35 Fig. 3 shows a multi-well assay plate structure

according to a second embodiment of the present invention;

Fig. 4a shows a third embodiment of a disc-style

WO 98/15356 PCT/GB97/02708

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st rue ture for conducting multi- tests;

Fig. 4b shows an enlarged cross-sectional detail of
Fig. 4a to allow snap-fitting of the plates in the disc
sectors ;

5 Fig. 4c is a fourth embodiment of a disc-style

structure for conducting multi-tests;

Fig. 4d shows a modification of the outer disc with
hinged sectors and which is applicable to the previous
embodiments ;

10 Fig. 5 depicts chemical/biochemical assay apparatus

for conducting an assay on reactions carried out using the
multi-well assay plate structures shown in Fig. 3 or Figs.
4a b, c and d, and

Figs. 6a and 6b depict the data and graphs

15 respectively of antigen/antibody biochemical assays carried

out using the apparatus of Fig. 5 on the assay plate shown
in Fig. 4a, b, c and d.

Reference is first made to Fig. 1 which shows a multi-
well assay plate, generally indicated by reference numeral

20 10, having a box-like construction with a rectangular

cross-section. The assay plate 10 comprises an upper plate
12, a lower plate 14, and side and rear spacers 16,18,20
all of which are made of a transparent polycarbonate. The
front of the box, indicated generally by the reference

25 numeral 22, is open to the surrounding space.

The spacers 16,18,20 are dimensioned to produce a
space 21 of uniform spacing d between the opposed inner
surfaces 12a, 14a of the upper and lower plates 12,14.
Spacing d is chosen such that a selected liquid is able to

3 0 flow through the space 21 between the upper and lower

plates 12,14 in a controlled manner by capillary or
capillary-like action. Generally, d is less than 0.5mm.

A small opening 23 extends through the upper plate 12
to communicate the inner space 21 with the exterior space

35 surrounding the container. Opening 23 is located close to

the rear wall 20 in order to prevent air-locks forming in
the container during filling as will be described in more

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PCT/GB97/02708

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detail below.

A regular array of wells or depressions 24 are formed
in the upper surface 14a of the lower plate 14. Typically,
the polycarbonate assay plate with wells 24 is produced by
5 suitably moulding the lower plate 14 or by etching or

pressing. The wells 24 are 2mm in diameter and 1mm deep
and typically have a volume of 5/xl and any suitable number
of wells may be provided. The wells are spaced 4mm apart
(centre to centre) .

10 Figs. 2a to 2c illustrate the process by which the

wells 24 of the assay plate 10 are filled with a liquid
analyte 25. The end 26 of a syringe 28 containing the
liquid analyte 25 is pressed into the opening 23 provided
in the upper plate 12 of the container 10 (Fig. 2a) so as

15 to form an air-tight seal between the periphery of the

syringe and the inner surface of the opening 23 . The
plunger 3 0 of the syringe 28 is then depressed to force the
liquid 25 through the opening 23 into the space 21 within
the plate 10. As best seen in Fig. 2b, due to the

20 capillary or capillary like flow of liquid through the

space 21, the entire space 21 is filled and wells 24 are
covered before liquid 2 5 begins to flow through the front
open face 22 of the container 10. When it is observed that
all of the space 21 is filled and the wells 24 are covered

25 with liquid, and preferably prior to liquid flowing out

through the front face 22, the plunger 30 of the syringe 28
is withdrawn. This action empties the space 21 of liquid,
but results in the wells 24 being filled with liquid 25 as
shown in Fig. 2c. Fig. 2d shows an enlarged cross-

30 sectional view through part of the assay plate structure

and showing how liquid is retained in wells 24 up to the
meniscus. As with the filling process, liquid flows from
the space 21 in a controlled manner. No puddles or drops
of liquid remain in the space 22, other than in the wells

35 24 .

It will be appreciated that prior to introducing the
liquid analyte 25 into the space 21, for example during the

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raanufacture of the assay plate 10, the wells 2 4 of the
plate 10 may be coated with an appropriate reactant . For
example, if it is desired to conduct antigen-antibody
reactions, the wells 24 are coated with an antigen. The

5 remainder of the surface 14a is coated with a blocking

agent to prevent antigen and antibodies from binding to
surface 14a. Once the wells 24 have been filled with the
liquid analyte 25, any antibodies present in the liquid
analyte 25 will bind with the antigens contained in the

0 wells 24. There is no binding of the antibodies to surface

14a. If it is necessary to conduct a further reaction in
the wells 24, e.g. to bind a coloured or fluorescent label
to the bound antibodies or exposed antigens, it is possible
to repeat the steps of Figs. 2a to 2c in order to introduce

5 the labelled components into the wells 24. Prior to

introducing the labelled components, if it is necessary to
rinse the wells 24 and the inner surfaces 12a, 14a of the
plate 10, this is again easily achieved by repeating steps
2a to 2c with the syringe 28 containing, for example,

0 distilled water.

There is illustrated in Fig. 3 a second embodiment of
the present - invention which depicts a multi-well assay
plate in the form of a disk 32 designed for use with a
rotating scanning device having a CD player type format.

5 One such device is described for example in WO96/09548.

The disk 3 2 shown in Fig. 3 comprises a pair of upper and
lower circular plates 34,36 sandwiched together to provide
a cylindrical space 38 therebetween. This space 38 is
divided into eight sectors 40 by radially extending spacers

0 42. A plurality of wells 44 are provided in each sector 40

(one set of which is shown in broken outline) by forming
the upper surface 36a of the lower circular plate 36 as
described with reference to Fig. 1. The wells 44 are of
the same size and are spaced as for Fig. 1.

5 Each sector 40 provides a chamber or space 4 6 which

can be filled independently via openings 48 provided
through the top surface of each sector 40. The peripheral

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edge 5 0 of each sector 4 0 is open to the surrounding space
to provide a vent for the sector 4 0 to allow liquid to flow
through the space or chamber 4 6 by displacing air
therefrom .

5 In order to enable the disk 32 to be compatible with

scanning devices such as are described in WO 96/09548, the
upper and/or lower plates 34,36 are made of transparent
polycarbonate to enable a light beam to be scanned across
the disk surface. The disk 32 is provided with a central
10 hole 52 to enable the disk 32 to be mounted on a rotatable

shaft .

As is described in WO 96/09548, one of the surfaces of
the upper or lower plates 34,36 may be provided with
digitally encoded address information which can be read by

15 the scanned light beam. This information may be encoded by

way of "pits" and "lans" pressed or moulded into one of the
plates. This address information can be used to provide
accurate location information on the part of the disk which
is begin scanned by the light beam.

20 There is shown in Fig. 4 a third embodiment of a disk

assay plate 54 which comprises upper and lower circular
transparent polycarbonate plates 56,58 which are spaced
apart by a number of radially extending spacer walls 60 to
create a plurality of disk sectors 62. The inner surfaces

25 56a, 58a of the circular plates 56,58 are both planar.

Each disk sector 62 is arranged to receive a sector
plate insert 64 which is a transparent polycarbonate plate
with a detachable handle 66 on the outer side to facilitate
entry and removal of the plate insert 64 in the sector 62 .

30 The plate insert 64 and spacer wall 60 have respective

recesses/projections (not shown in the interest of clarity)
which allow the assay plate 64 to be inserted only in the
correct orientation. The plate 64 has a groove 68, as
shown in Fig. 4b for example, which allow the insert to be

35 snap-fitted over a projection 70 upstanding from plate 58

into the sector. The thickness of the sector insert plate
64 is marginally less than the spacing provided between the

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upper and lower plates 56,58 so that the plate insert 64
can be pressed/fitted into one of the disk sector 62 to
define a liquid receiving chamber or space 73 between the
upper surface 64a of the insert plate 64 and the lower
5 surface 56a of the upper disk plate 56. Openings 72 are

provided through the upper plate 56 into each disk sector
64 whilst the space 70 between the radially outermost
peripheral edge 74 of the insert plate 64 and the upper
plate 5 6 provides a further vent or filling opening into

10 the disk sector 64.

The surface 64a of the insert plate 64 is provided
with a plurality of wells 76 as described with respect to
Fig. 1. The wells are 2mm in diameter, 1mm in depth and
4mm apart (spaced between centres) . These wells are filled

15 by introducing liquid into the disk sector 64 through the

upper opening 72 to fill space 70 and subsequently
withdrawing the liquid through the same opening as
previously described.

Reference is now made to Fig. 5 of the drawings which

20 depicts assay apparatus for conducting an assay on

reactions carried out using the assay plate structures of
the already described embodiments. However, for

convenience, the assay apparatus will be described in
combination with the preferred embodiment shown in Figs.

25 4a, b with like numerals referring to like parts.

In this case the plate 54 is mounted on a shaft 74
carried by a turntable 77. The apparatus includes a

suitable automatic fluid filling/withdrawal system,
generally indicated by reference numeral 80, which operates

30 a syringe 82 to dispense/retrieve fluid from a reservoir 84

via the openings 72 into the space 70 between the plate
surface 56a and the surface 64a of each sector plate 64 .
The fluid can of course be dispensed and retrieved manually
if desired. This 'is achieved for each sector by rotating

35 the disk plate 54 to a suitable position to allow fluid

filling/withdrawal. It will be appreciated that the

plates are pre-prepared with various reagents, e.g.

WO 98/15356 PCT/GB97/0T708

-15-

antigens, and they are inserted in the appropriate wells
76, as described with reference to Figs. 4a, 4b. The
plates are first flooded with fluid carrying antibodies and
withdrawal of the fluid leaves the antibody/antigen
5 reagents filling the wells 76 resulting in a reaction.

The following example of an assay within the
embodiment shown in Fig. 4b is described to provide a
better understanding of the steps involved:
Multi-Antigen Elisa Using Sectors
10 1. The underside of upper surface (56a) of is coated with

silicone spray to aid fluid movement. Sector plates

64 are also coated including wells 76. Any excess

silicone is removed.

2. Sectors wells 76 are loaded by hand with a panel of
15 seven antigens - Human Serum Albumin, Antitrypsin,

Macroglobulin, Antithrombin III, Catalase,

Antichymotrypsin and Plasminogen at a concentration of
20ug/ml in PBS and a volume of 2ul/well . Control
wells contain PBS only. Antigens can be arranged in
20 blocks of the same on the sector plate 64 in a series

giving a panel of tests evenly distributed over the
sector. Incubate at room temperature for 15 minutes.

3. Wash with 0.05% PBS-Tween using flood/fill technique -
lml is flooded across the sector plate via holes 72 in

25 the top plate using a lml pipette. This pipetted up

and down three times then withdrawn and the washing
discarded. This repeated a further three times to
ensure complete washing.

4 . Blocking is carried out to prevent reactions occurring
30 other than at well sites with 50mg/ml Bovine Serum

Albumin (BSA) (in PBS) using flood/fill. lml of

BSA/PBS is flooded across the sector, pipetted up and
down three times, withdrawn and discarded. This
allows all wells 76 to be filled simultaneously.
35 Incubate for 15 minutes at room temperature.

5. Wash as before.

6. Primary antibodies are applied to the sector plate 64

WO 98/15356 PCT/GB97/02708

-16-

as a mixture using flood/fill with each individual
antibody at the following concentrations: anti-Human
Serum Albumin 1/1000, ant i -Antitrypsin 1/2000, anti-
Macroglobulin 1/2000, anti-Antithrombin III 1/1000,
5 anti-Catalase 1,1000, anti-Antichymotrypsin 1/1000,

anti- Plasminogen 1/1000. Antibodies are diluted in
0.5mg/ml BSA/PBS. Incubate for 10 minutes at room
temperature .
7. Wash as before.

10 8. Second antibody is Amdex anti-IgG (peroxidase

conjugate) at a concentration of 1/1000 in 0.5 mg/ml
BSA/PBS. After washing this is applied to the sector
using flood/fill . Incubate at room temperature for
10 minutes.

15 9. Wash as before.

10. The substrate is insoluble Tetramethylbenzidine (TMB) .
This reacts with the peroxidase on the second antibody
to produce an intense blue colour. After washing
this is applied to the sector plate 64 by flood/fill

20 but is left flooded across the sector plate 64 after

pipetting up and down several times. Incubate for 10
minutes at room temperature.

11. Remove TMB and discard. Wash out the wells with
distilled water four times by flood/fill. A blue

25 precipitate will be evident in wells with a positive

reaction. No colour is produced in negative wells.
Store sections in dark as TMB will slowly fade in
daylight .

The data for the above assay is shown in Fig. 6a and
30 is graphically represented in Fig. 6b which is

reproducible and is representative of a large number
of experiments (712) .

It will be seen that there is a significant measurable
change for each antibody/ant igen reaction compared with the
35 background level. The reaction results in an optical

change, from transparent to coloured (blue) and which is
measured using an optical detector which measures light

WO 98/15356 PCT/GB97/02708

-17-

transmissivity through the disk and wells. in this case
optical assessment was carried out using the apparatus as
shown in Fig. 5 by locating the plate 64 in a light
transmissive microscope 80 (Zeiss Axiophot fitted with a
5 JVC video camera 83 (Model No. TK-1280E)) and sensing the

change in optical signal. The output of the video camera
is connected to Macintosh IICx 85 with video frame capture.
The results can be displayed via the Mac display 87 or" a
hard copy provided by printer 86. Analysis was carried

10 out by measuring mean grayscale values in centre of wells

quantified by NIH Image software. Background levels taken
from sectors which had not been exposed to immuno- chemicals
or chromogen were subtracted from all experimental wells.
Experimental wells contained array or seven separate

15 antigens listed above. In addition, experimental controls

were carried out in which specific antigen was omitted
wells and wells exposed to the same regime of blocking,
antibody binding and exposure to chromogenic substrate .
The average reading from these experimental controls minus

20 mean reading from the sector alone was defined as the

background level of staining. Experimental readings from
the seven .specific antigens providing signals of
approximately five to six times greater than this
background. It will be observed that there is no cross-

25 contamination between wells 76 because of the efficiency of

withdrawal and because the substrate in this case is
insoluble. However, this assay would also work

satisfactorily for soluble substrates because of fluid
withdrawal from the sector plate 64 leaving fluid in the

30 wells 76 only, not on surface 64a.

In a modification, if it was unnecessary to withdraw
all of the liquid to leave a film on surface 64, the assay
would still work with an insoluble substrate in each well;
cross-contamination would still not occur. However, this

35 arrangement would be unsatisfactory for soluble substrates

in the wells as the film could cause dispersal to other
locations and provide contamination of other wells.

WO 98/15356 PCT/GB97/02708

-18-

With the embodiment shown in Figs. 4a, 4b the disk
sector plate 54 is more suitable for conducting a variety
of different assays, e.g. antigen/antibody assays for
different patients, i.e. one pat ient /sector .
5 It will be appreciated that modifications may be made

to the above described embodiments without departing from
the scope of the present invention. For example, the
opening through which the liquid analyte is introduced may
be provided through the lower plate of the mult i -well

10 container. More than one opening can be used for faster

flooding. This opening may be arranged to receive the tip
of a syringe needle. The vent opening may also be provided
in any one of the walls of the container although it is
preferably provided in a peripheral wall. The opening 22

15 may be provided by a single opening 22 or by a series of

openings or vents as shown in Fig. 4d for example. A
laser may be used with CD optics instead of the microscope
and video camera for the embodiment of Fig. 4. The top
plate in the embodiment of Figs. 3 and 4 may be snap-fitted

20 to the lower plate and may be snap- fitted onto a CD base

plate which would receive sections and provide the
advantage of positioned information. As shown in Fig. 4c
the upper planar surface 56 can have sector covers
connected to a lower surface or central boss by a hinge,

25 for example an integrated living hinge 90 at the inner

radius to allow each disk sector 62 to be pivotably raised
and lowered and allow sector plates 64 to be inserted into
each sector. The well size and spacing may be varied as
required, for example the wells could be 3mm in diameter;

30 l.5mm apart and spaced 5.5mm between centre. The exact

size and spacing is a matter of choice consistent with the
requirement that fluid is retained in the wells after
withdrawal as described above. However, the wells could
also be filled during flooding of the space depending on

35 the well size, type of plastic and fluid properties.

However, liquid will still be retained in the wells upon
withdrawal of the liquid. Also, the structure and inserts

WO 98/15356 PCT/GB97/02708

-19-

made may be of any suitable optically transmissive plastic,
such as polystyrene or perspex (TM) . The handle 66 may be
integrated with or detachable from plate 64 . As shown in
Fig. 4a the radially extending ribs may have radial
5 shoulders 92 to define a recess 94 for receiving the plate

64 also defining the spacing height between the surface 64a
of the plate 64 and the underside 56a for receiving the
liquid. Suitable materials may be used to coat the

interior of the sectors to aid fluid movement as described

10 with reference to silicone above. This may be applied to

the underside of the top surface and to the top surface of
the plate as for the other embodiments. Suitable
materials may be used to increase the hydrophobicity of
liquid across the sector and hydrophilicity to and movement

15 of liquid into the desired locations, e.g. wells. The

wells may be coated with a suitable optical reflective
material to enhance the reflection of light and observation
of reactions occurring within the wells and, similarly,
lenses may be located in the top or bottom light

20 transmissive plate to improve optical assessment of the

reaction. These lenses may be moulded into the upper or
lower plates during the manufacture as is well known in
plastic moulding processes. Separate optical elements may
be used instead, if appropriate.

25 In a modification to the embodiments described, the

wells are absent from the upper surface of the plate and
that plate retains its planar surface to enable a thin,
uniform layer of liquid to be introduced into the space
between the upper disk plate and the insert plate. An

30 insoluble substrate with reagent or reagents (e.g. an

antigen) may be applied directly to the planar surface of
the insert plate by for example applying spots of reagent
thereto .

For certain applications, it may be appropriate to
35 provide each insert with a lid which can be slid into the

space between the insert and the upper plate 22 of the disk
following filling of the wells. The lower surface of the

WO 98/15356 PCT/GB97/02708

-20-

lid may be arranged to be flush with the surface of the
insert so as to close off each well. This prevents liquid
from being thrown out of the wells during spinning of the
disk during automated reading and analysis. The
5 invention has use in immunoassay applications including

tests for sexually transmitted diseases, parasites,
allergens, cancer markers and cardiac markers, either in
laboratories or at point-of -care locations, for example
medical practitioners offices or the like. Other

10 applications of the invention are in chemical and

biochemical assays. Examples of such assays include

immunoassay, clinical biochemistry tests, nucleic acid
analysis and receptor ligand interactions. Examples of
clinical biochemistry uses would be in measurement of serum

15 analytes such as glucose, urea, creatinine and enzymes such

as alkaline phosphatase. Immunoassay application include
tests designed to detect infectious organisms, viruses,
parasites as well as endogenous analytes such as
circulating hormone levels and cancer markers. Examples

2 0 of chemical analysis include measure of phosphate and

nitrate levels in water, environmental and industrial
monitoring including potable and waste water and process
monitoring. The system could be used in a variety of
settings including clinical laboratories, doctor's and

25 veterinary surgeries as well as industrial and research

laboratories .

* «

WO 98/15356 PCT/GB97/02708

-21-

CLAIMS

1. A mult i -well assay plate structure comprising:
a first upper surface,

a second lower surface having a plurality of wells
disposed therein,
5 the first and second surfaces defining a chamber

having an inlet and an outlet, the inlet and outlet
allowing fluid to be introduced and withdrawn from the
chamber, the wells being proportioned and dimensioned to
retain a volume of fluid in each well following withdrawal
10 of the liquid.

2 . An assay plate structure as claimed in claim 1 wherein
the chamber is shallow enough to allow fluid to fill the
wells and the chamber.

3. As assay plate structure as claimed in claim 1 or 2
15 wherein the wells are deep enough to retain a volume of

fluid following withdrawal of fluid in the space above the
wells .

4 . As assay plate structure as claimed in any preceding
claim wherein the plate structure is sector- shaped with a

20 handle at the longer arc-portion to facilitate locating the

sector on a disc .

5. An assay plate structure as claimed in claim 4 wherein
a plurality of sector- shaped plate structures are carried
by a disc.

25 6 . An assay plate structure as claimed in claim 5 wherein

also the sectors and discs are made of plastic and the
sectors can be snap-fitted onto the disc.

7 . An assay plate structure as claimed in claim 5 or 6
wherein the sectors and the disc include lock and key

30 portions to allow the sectors to be snap- fitted in the

correct orientation only.

8 . As assay plate structure as claimed in any one of
claims 1 to 4 wherein the assay plate structure is a disc
moulded in one piece with a plurality of wells.

35 9. An assay plate structure as claimed in claim 8 wherein

the structure includes an upper disc with a plurality of

WO 98/15356 PCT/GB97/02708

-22-

hinged sectors for allowing the sector to pivot from and
return to the plane of the disc to facilitate the insertion
and removal of assay plates in the sector space.

10. An assay plate structure as claimed in any preceding
5 claim wherein the disk structure has a circumferential

gutter extending around its periphery to facilitate
collection of fluid following fluid withdrawal from the
chamber .

11 . An assay plate structure as claimed in any preceding
10 claim wherein the plate structure and sector inserts are

made of optically transmissive plastic.

12. A multi-well assay structure comprising an upper
surface and a lower closely spaced opposed surface, said
upper and lower surfaces defining a relatively shallow

15 space therebetween, the lower surface having a plurality of

wells therein, at least two spaced apart openings providing
access to said space from an external location, wherein a
fluid introduced into said space through one of said
openings substantially fills the space and covers all of

20 the wells and said fluid when subsequently withdrawn

through the same or the other opening leaves the wells
substantially filled with liquid.

13. An assay plate structure as claimed in claim 12
wherein the spacing between said upper and lower surfaces

25 is sufficiently small to facilitate the flow of fluid in

said space by capillary or capillary like action.
14 . An assay plate structure as claimed in claim 13
wherein the spacing is less than 1mm.

15. An assay plate structure as claimed in claim 14
30 wherein the spacing is less than 0.5mm.

16. An assay plate structure as claimed in claims 13, 14
or 15 wherein the surfaces are provided by respective upper
and lower plates which are spaced apart by one or more
spacer walls.

35 17. An assay plate structure as claimed in any one of

claims 13 to 16 wherein the opening through which fluid is
introduced into said space is provided through either the

WO 98/15356

-23-

PCT/GB97/02708

uoper or lower surface.

T An assay plate structure as claimed in claim 17
Irein the opening is provided through the upper surface^
19 An assay plate structure as claimed in. any one of
claims 13 to 18 wherein said opening for introducing a
fluid comprises a relatively small opening arranged to
r ceL Z end of a syringe or similar liguid injecting,
device, where the opening forms a substantially axr-txght
qpa i around said end.

20 An assay plat. «™ t «. a. claim.d in any one.
claims 12 to 19 wherein the underside of said upp.r surface
TZ contains, and th. upp.r surface of
crested to incr.asa th. hydrophobic^ of saoh surfaces.
" In assay plate structure as claimed in any on. of
claims 12 to 20 wherein th. clti-w.ll structure as a desk
^ch comprises upper and loser circ ular pi at.s, the

internal surface, of which respectively define said upp

=»r,rl lower opposed surfaces.

2 " T assly Piste structure as claimed in claim 21
ILZ the second openin g is provided at th. p.rrph.rsi

T » structure as claimed in claim 21 or 22

I' .Tn the space between th. upp.r and lower plates is
bdlvided by =ne or more dividing walls, to provide a
Tlural " of Ilti-well Plates in which case each space is
Sovtoee with an op.ning and a vent to enable each space to

:i 'r^^ateltraoture as oiaimed in claim 23

rz trss ~ rsrr: o, -

claim7ll to 2/wh.r.in at least on. of th. upp.r and lower
; . Z forming the structure are to en b e

26 . An assay pi plates may

— rrefl ^ .."o^aC radiation entering
"to the struccur. through th. transp.r.nt plat.

WO 98/15356 PCT/GB97/02708

-24-

transverses the structure in both directions for providing
improved signal detection.

27. An assay plate structure as claimed in any one of
claims 12 to 2 0 wherein there is provided a disc arranged

5 to receive a plurality of sector-shaped inserts each of

which comprises a generally planar upper surface having a
plurality of wells provided therein, the disk having, for
each insert, a substantially planar surface arranged, in
use, to oppose said substantially planar insert surface and
10 means for retaining the insert in position so that the

respective planar surfaces are in a closely spaced
arrangement to one another, and to said at least two
openings .

28. An assay plate structure as claimed in claim 27

15 wherein the opening for filling the space is provided

through the planar surface of the disc.

29. An assay plate structure as claimed in claim 27 or 28
wherein the vent opening is provided at, or adjacent to,
the peripheral edge of the disc.

20 30. An assay plate structure as claimed in claims 27 to 29

wherein the disc comprises upper and lower circular plates
separated by radially extending spacers.

31. An assay plate structure as claimed in claim 30
wherein said planar surface of each insert comprises

25 upstanding walls around at least a portion of its periphery •

for the purpose of sealing the inner edges of the insert to
the opposed planar surface of the disc, thereby to prevent
seepage of liquid around the insert .

32. A method of filling the wells of the multi-well
3 0 structure of the above first aspect of the present

invention, said method comprising the steps of:

introducing a fluid into said chamber through one of
said openings to substantially flood the chamber;

and subsequently withdrawing the fluid from the
3 5 chamber through the same or the other opening to leave

liquid in the wells.

33. A . method as claimed in claim 8 wherein the method

WO 98/15356 PCT/GB97/02708

-25-

further includes the step of forming an air tight seal
between the fluid inlet and an end region of a syringe or
similar liquid injecting device, and injecting fluid
through the opening into the chamber and subsequently
5 sucking liquid out of the space through the opening.

34 . A method conducting a chemical or biochemical assay
said method comprising the steps of :

providing a surface within a substantially enclosed
chamber having a plurality of wells at spaced locations
10 sufficient to allow monitoring of a reaction at each well

location,

treating each well with a first reagent, flooding the
enclosed chamber and covering the wells with a fluid
carrying at least a second reagent,
15 removing excess fluid from said chamber to leave a

mixture of said first and second reagents in each well, and
optically assessing each well and determining if a
reaction occurred and correlating the reaction results to
provide an assay of the chemical or biochemical reactions
20 under test.

35. A method as claimed in claim 34 wherein the step of
optical assessment is carried out automatically using
optical reading apparatus.

36. A method as claimed in claim 34 or 35 wherein the
25 surfaces with wells having the first fluid carrying

reagents are prior prepared for loading into the structure.

37. A method as claimed in any one of claims 34 to 36
wherein after optical assessment of the results of the
assay, the automated fluid handling apparatus is used to

3 0 inject and withdraw rinsing fluid a predetermined number of

times from the well tray to clean the wells for receiving
subsequent samples for assay.

38. Chemical/biochemical assay apparatus comprising an
assay plate structure defined in said first aspect and

35 having a plurality of wells for receiving samples to be

assayed,

fluid handling means for introducing and removing

WO 98/15356 PCT/GB97/02708

-26-

fluid reagents into said assay plate structure to allow a
fluid reagent mixture to be retained in each well, and

optical assessment means for measuring optical result
of the reaction in each well.
5 39. An assay plate structure for use in conducting optical

assays of a fluid analyte, the plate structure comprising:

a disc for rotation about a central axis, the disc
having upper and lower plates and a plurality of
substantially radially extending walls disposed between the
10 plate, wherein said walls sub-divide the disc into a

plurality of disc sectors; and

a plurality of disc inserts arranged to be received by
respective disk sectors and to be retained therein,

the structure further a plurality of openings through
15 the upper surface, at least one opening above each disc

sector for introducing a liquid analyte into the sector
space between the plate and the disc insert.

40. An assay plate structure as claimed in claim 39
wherein the disc further comprises a lower plate, spaced

20 apart from said upper plate by said radially extending

walls .

41. An assay plate structure as claimed in claim 40
wherein the upper surface of each disc insert and the
opposed surface of the plate are substantially planar.

25 42. An assay plate structure as claimed in any one of

claims 3 9 to 41 wherein a vent opening is provided for each
disc segment around the periphery thereof, between the
radially outer edge of the upper plate and each disc
insert .

30 43. A multi-sample assay plate structure comprising:

a first upper surface,

a second lower surface spaced from the upper surface
by wall means to define a chamber with the upper and lower
surfaces spaced a preset distance apart,
35 the chamber having an inlet and an outlet, the inlet

and outlet allowing fluid to be introduced to, and
withdrawn from, the chamber, the lower surface being

WO 98/15356 PCT/GB97/02708

-27-

adapted to receive spots of an insoluble substrate,
carrying a first reagent, or no reagent if a control spot,
to create a plurality of separate reaction sites, such at
least a reagent is present in the fluid for reacting with
the first reagent to create an observable reaction in the
chamber .

44. A method of conducting an assay using the structure of
claim 43 including the steps of,

disposing a plurality of spots of an insoluble
substrate on said lower surface a predetermined distance
apart to create a plurality of reaction sites, said spots
carrying a first reagent, or none if a control spot,

flooding the chamber with fluid carrying at least one
second reagent, withdrawing the fluid from the chamber to
leave sufficient spots of fluid in contact with the
substrate spots, and

optically monitoring each spot location to detect a
reaction .

WO 98/15356

PCT/GB97/02708

Fl G.3

SUBSTITUTE SHEET (RULE 26)

WO 98/15356

PCT/GB97/02708

2/3

SUBSTITUTE SHEET (RULE 26)

WO 98/15356

3/3

PCT/GB97/02708

,81

87 v

Fl G.5

klilii

V N \\\\

tw iiuTn^agent Seven anti getLassay.

Date plastic +Btocking agent

♦ ♦

76.4 80.2 106.6 108.8 103.3 103.4 102.2 103.6 1088

76.4 85.5 106.4 103.9 101.3 102.7 98 5 111.2 109

76.83 81.4 105.4 103.1 105.2 104? 101 106 2 1095

74.53 81.6 1065 104.7 104.7 106.4 100.7 1029 1064

78.62 82.6

76.75 79.6

77.6 78.9

77.8 833

Mean-bk ad 5 29.4 283 269 27.4 23.8 292 31.6

St.Dev 0.56 2.44 0.56 2.44 1.75 1.60 1.54 3.76 1.38

F I G.6a

Key

1 = Background

2= Human serum albumin

3 = Antitrypsin

4= Macroglobulin

5= Antithrombin III

6= Catalase

7= Antichymotrypsin

F I G.6b

SUBSTITUTE SHEET (RULE 26)

INTERNATIONAL SEARCH REPORT

national Application No

PCT/GB 97/02708

A, CLASSIFICATION OF SUBJECT MATTER

IPC 6 B01L3/00 G01N33/543

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system I otto wed by classification symbols)

IPC 6 BOIL G01N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category • Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

EP 0 430 248 A (MOCHIDA PHARM CO LTD) 5
June 1991

see page 3, line 34-38

see page 11, line 10 - page 13, line 8;

figures 17,18

US 5 424 036 A (USHIKUB0 MASAO) 13 June
1995

see column 4, line 57 - column 5, line 52;
figures 2,4,5

US 5 281 540 A (MERKH CARL W ET AL) 25
January 1994

see column 4, line 34-68
see column 6, line 15-47
see column 9, line 50-61; figures 1,2,5

-/—

43,44

Further documents are listed In the continuation of box C.

Patent family members are listed in annex.

4 Special categories of cited documents :

"A" document defining the general state of tha art which is not
considered to be of particular relevance

"E" earlier document but published on or after the international
filing date

"L" document which may throw doubts on priority claim(s) or
which Is cited to establish the publication date of another
citation or other special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or
other means

"P" document published prior to the intemationai filing date but •
later than the priority date claimed

"T" later document published after the international filing date
or priority date and not In conflict with the application but
cited to understand the principle or theory underlying the
invention

"X" document of particular relevance; the claimed Invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document Is taken alone

"Y" document of particular relevance; the claimed Invention

cannot be considered to involve an inventive step when the
document Is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.

document member of the same patent family

Date of the actual completion of the international search

27 February 1998

Date of mailing of the international search report

10/03/1998

Name and mailing address of the ISA

European Patent Office, P.B. 581 8 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040. Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016

Authorized officer

Zinngrebe, U

Form PCT/1SA/21 0 (second shoot) (July 1992)

page 1 of 2

INTERNATIONAL SEARCH REPORT

i national Application No

PCT/GB 97/02708

C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category ■' Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US 4 077 845 A (JOHNSON LEIGHTON CLIFFORD)

7 March 1978

see column 6, line 3-18

see column 6, line 23-47

US 4 195 060 A (TERK HAROLD S) 25 March
1980

see column 3, line 5-68

GB 1 572 596 A (OPTO ELECTRONIC DISPLAYS

LTD) 30 July 1980

see page 2, line 43-109; figure 1

US 5 338 666 A (M0NTH0NY JAMES F ET AL)
16 August 1994

see column 6, line 55 - column 7, line 9
see column 7, line 30-66

1,12,32,
34,38

Form PCT/lSA/210 (continuation of second shoot) (July 1992)

page 2 of 2

INTERNATIONAL SEARCH REPORT

Information on patent family members

I lationai Application No

PCT/GB 97/02708

Patent document
cited in search report

Publication
date

Patent family
member(s)

Publication
date

EP 0430248 A

05-06-91

642444

21-10-93

6702690

06-06-91

2031001

31-05-91

3223674

02-10-91

5147607

15-09-92

US 5424036 A 13-06-95

US 5281540 A 25-01-94

5297007

12-

11-

-93

4313399

28-

10-

-93

5075077

24-

12-

-91

2123785

23-

■05-

-93

0649534

26-

■04-

-95

7501149

02-

-95

9310454

27-

■05-

-93

137025

15-

•05-

-96

625064

02-

07-

-92

3884889

08-

■02-

-90

1335345

25-

■04-

-95

68926272

23-

■05-

-96

68926272

19-

•12-

-96

0353590

07-

•02-

-90

2088878

01-

■10-

-96

2075954

15-

•03-

-90

US 4077845 A 07-03-78

US 4195060 A 25-03-80

504980

01-

11-

•79

3145577

21-

•06-

•79

1090239

25-

11-

•80

2817145

26-

■10-

•78

2388046

17-

•11-

•78

1548530

18-

•07-

•79

1130914

17-

■01-

•83

53144186

15-

•12-

•78

57024749

26-

•05-

•82

4178345

11-

12-

-79

522647

17-

■06-

-82

4388879

16-

■08-

•79

1122890

04-

•05-

-82

2904597

30-

-08-

-79

50879 A,B,

09-

•08-

•79

Form PCT/ISA/210 (patent family annex) (July 1992)

page 1 of 2

INTERNATIONAL SEARCH REPORT

Information on patent family members

I lational Application No

PCT/GB 97/02708

Patent document
cited in search report

Publication
date

Patent family
member(s)

Publication
date

2417109

07-09-79

2014307 A.B

22-08-79

1164381

26-08-83

54115187

07-09-79

57054746

19-11-82

7900987

10-08-79

448029

12-01-87

7900854

09-08-79

874007

07-08-79

7900376

30-01-80

US 4195060 A

GB 1572596 A

30-07-80

NONE

US 5338666 A 16-08-94

5182082 A

26-01-93

137798 T

15-05-96

640838 B

02-09-93

8965791 A

30-07-92

69210424 D

13-06-96

69210424 T

05-12-96

0496200 A

29-07-92

2086556 T

01-07-96

2096899 C

02-10-96

4315946 A

06-11-92

8012135 B

07-02-96

240604 A

27-06-94

Form PCT/IS A/210 (patent family annex) (July 1992)

page 2 of 2

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